Structural modeling of dual‐affinity purified Pho84 phosphate transporter

Lagerstedt, Jens O.; Voss, John C.; Wieslander, Åke; Persson, Bengt L.

doi:10.1016/j.febslet.2004.11.012

Cited by 35 publications

(34 citation statements)

References 41 publications

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“…Pho84, a high-affinity Pi transporter in yeast, is internalized after phosphorylation and ubiquitination and is sorted into a vacuolar degradation pathway when Pi is resupplied (Lundh et al, 2009). Pho84 is composed of two bundles of six helical transmembrane segments connected by a long central loop (Lagerstedt et al, 2004). Interestingly, the deletion of the central loop abolished these posttranslational events, supporting the assertion that the ubiquitination and phosphorylation sites are gathered in this region (Peng et al, 2003;Lundh et al, 2009).…”

Section: Ubiquitination-mediated Endocytosis As a Crucial And Conservmentioning

confidence: 49%

NITROGEN LIMITATION ADAPTATION, a Target of MicroRNA827, Mediates Degradation of Plasma Membrane–Localized Phosphate Transporters to Maintain Phosphate Homeostasis in Arabidopsis

2013

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Section: Ubiquitination-mediated Endocytosis As a Crucial And Conservmentioning

confidence: 49%

NITROGEN LIMITATION ADAPTATION, a Target of MicroRNA827, Mediates Degradation of Plasma Membrane–Localized Phosphate Transporters to Maintain Phosphate Homeostasis in Arabidopsis

2013

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“…The Arabidopsis TAAC gene was PCR-amplified with oligonucleotides designed to exclude the first 177 base pairs of the gene (corresponding to the predicted transit peptide), cloned, and recombined as a fusion construct with an N-terminal hexahistidine tag followed by Xpress epitope and a C-terminal FLAG epitope (DYKD-DDDK) into a pTrcHisB plasmid for expression in E. coli. Such a set-up has previously been employed to purify another membrane-embedded protein (29). The detailed protocols are given under supplemental "Experimental Procedures.…”

Section: Methodsmentioning

confidence: 99%

Identification, Expression, and Functional Analyses of a Thylakoid ATP/ADP Carrier from Arabidopsis

Thuswaldner

Lagerstedt²,

Rojas-Stütz

et al. 2007

Journal of Biological Chemistry

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106

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TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.Chloroplasts perform oxygenic photosynthesis in algae and plants and have evolved by endosymbiosis from cyanobacteria. Chloroplasts have two distinct membrane systems, the double envelope surrounding the organelle and an internal membrane system named thylakoids. The envelope membrane represents the interface between the cytoplasm and chloroplast stroma, whereas the thylakoid membrane separates the stroma and the lumenal space. Altogether ϳ800 membrane proteins have been identified by proteomics in the envelope and thylakoid membranes of Arabidopsis thaliana (for reviews, see Refs. 1 and 2). As expected, the main function for the identified envelope proteins was transport of ions and metabolites, whereas photosynthesis was attributed to most of the identified thylakoid proteins. The major protein complexes in thylakoids are photosystems (PS) 4 I and II, the cytochrome b 6 f complex, and the proton-translocating ATP synthase. These photosynthetic complexes contain not only proteins but also pigments and other cofactors. Their assembly, activity, and removal require a large number of auxiliary, regulatory, and transport proteins (3, 4). Many biochemical reports pointed to the existence of transport activities in the thylakoid membrane, such as calcium transport (5), copper transport (6), anion channels (7), cation channels (8, 9), and nucleotide transport (10). Only the thylakoid copper transporter was identified at the genetic level in Arabidopsis (11). No hydrophobic proteins related to the above-mentioned transport activities were identified in the previous proteomic works on Arabidopsis thylakoid membranes (for a review, see Ref.2). Therefore, genetic strategies are required for identification and elucidation of their role in optimal function of the thylakoid.ATP is produced during the light-dependent photosynthetic reactions on the stromal side of the thylakoid membrane. Besides its utilization during CO 2 fixation in the stroma, ATP drives many energy-dependent processes in thylakoids, including protein phosphorylation, folding, import, and degradation.

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“…The radioactivity of this suspension was measured using liquid scintillation. (26) and structural alignment analyses (21)(22)(23)(24)(25), using as templates the X-ray structures of LacY (18) and GlpT (19), have suggested that the general fold of 12 TM MFS-transporters is conserved. However, the fact that sequence similarity is often very poor among proteins in this superfamily prohibits reliable structural alignment studies for many proteins, including the Mdr transporter MdfA.…”

Section: Mmol) An Aliquot Of 180 µL Of the Resinprotein-[ 3 H] Tppmentioning

confidence: 99%

3D Model of the Escherichia coli Multidrug Transporter MdfA Reveals an Essential Membrane-Embedded Positive Charge

et al. 2005

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MdfA is an Escherichia coli multidrug transporter of the major facilitator superfamily (MFS) of secondary transporters. Although several aspects of multidrug recognition by MdfA have been characterized, better understanding the detailed mechanism of its function requires structural information. Previous studies have modeled the 3D structures of MFS proteins, based on the X-ray structure of LacY and GlpT. However, because of poor sequence homology, between LacY, GlpT, and MdfA additional constraints were required for a reliable homology modeling. Using an algorithm that predicts the angular orientation of each transmembrane helix (TM) (kPROT), we obtained a remarkably similar pattern for the 12 TMs of MdfA and those of GlpT and LacY, suggesting that they all have similar helix packing. Consequently, a 3D model was constructed for MdfA by structural alignment with LacY and GlpT, using the kPROT results as an additional constraint. Further refinement and a preliminary evaluation of the model were achieved by correlated mutation analysis and the available experimental data. Surprisingly, in addition to the previously characterized membrane-embedded glutamate at position 26, the model suggests that Asp34 and Arg112 are located within the membrane, on the same face of the cavity as Glu26. Importantly, Arg112 is evolutionarily conserved in secondary drug transporters, and here we show that a positive charge at this position is absolutely essential for multidrug transport by MdfA.

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Structural modeling of dual‐affinity purified Pho84 phosphate transporter

Cited by 35 publications

References 41 publications

NITROGEN LIMITATION ADAPTATION, a Target of MicroRNA827, Mediates Degradation of Plasma Membrane–Localized Phosphate Transporters to Maintain Phosphate Homeostasis in Arabidopsis

NITROGEN LIMITATION ADAPTATION, a Target of MicroRNA827, Mediates Degradation of Plasma Membrane–Localized Phosphate Transporters to Maintain Phosphate Homeostasis in Arabidopsis

Identification, Expression, and Functional Analyses of a Thylakoid ATP/ADP Carrier from Arabidopsis

3D Model of the Escherichia coli Multidrug Transporter MdfA Reveals an Essential Membrane-Embedded Positive Charge

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